Monoclonal antibodies and antibody domain-based molecules constitute the majority of protein therapeutics under clinical investigation (1, 2). Monoclonal antibodies are potent in a diverse range of therapeutic indications, and are easily generated. The specificity of the antibody is primarily determined by sequences in the CDRs located in the variable domain. The process of selecting a clinical candidate starts with screening for functional properties of a large number of monoclonal antibodies. Screening is followed by detailed in vitro profiling of up to hundreds of molecules to identify a monoclonal antibody that fulfills all desired functional criteria and additionally displays chemical and biophysical stability. Hence, monoclonal antibodies with instability issues that can affect their structure and biological function have to be identified and deselected to avoid unwanted degradation during manufacturing and storage, and in vivo after application.
One degradation reaction occurring in proteins is the chemical degradation of asparagine (Asn) (3) and aspartate (Asp) residues (4, 5). These reactions may be kept under control by appropriate formulation conditions (6-9). If Asn and Asp residues are involved in antigen recognition, their chemical alteration can lead to a reduction of potency of the antibody (10-14).
Diverse parameters were proposed which may influence the degradation propensity of Asn and Asp residues, e.g. the primary sequence (3, 5, 16, 33, 40, 51-56), the solvent dielectric constant, temperature, and the pH, mostly in the peptide (52, 53, 57-59), but also in the protein context (7, 10, 35, 60). Already in the 1980s, several structural requirements were suggested as principal determinants for protein deamidation (5, 61) which have later been confirmed and extended (34, 37, 38, 40, 46, 51, 62-64).
Despite accumulated knowledge about the degradation mechanism and its environmental requirements, spontaneous deamidation and isomerization in monoclonal antibodies remains an unresolved issue.